The brain is arguably the most complex organ in higher mammals in terms of diversity of resident cell types, anatomical organization, and intricate modes of intercellular communication and protein regulation. Each of these factors presents a tremendous technical challenge for researchers interested in applying large-scale biological approaches to the study of brain physiology and pathology. Proteomics, for instance, is most adept at mapping protein expression and post-translational events in biological samples enriched in a single cell type (e.g., cultured cells or relatively homogenous organs, such as liver). The brain, however, offers a much more complicated scenario. Indeed, brain disorders such as drug dependence and withdrawal may be due to changes in the molecular composition and function of only a small number of neurons or neuronal circuits dispersed among a large background of unaffected cells. Neuronal plasticity is furthermore known to rely heavily on post-translational events that regulate protein structure and activity, often within specific subcellular compartments (e.g., the synapse). Detecting and quantifying such 'rare'biochemical events is beyond the capacity of contemporary proteomics methods. As a result, the tremendous opportunity afforded by proteomics to discover new proteins and pathways that contribute to higher-order brain function has, to date, gone unrealized. This proposal addresses this Grand Opportunity (GO) through creation of the first proteomics platform for quantitative analysis of protein expression and post-translational modification in the mammalian brain. This platform, termed qPEMM (for quantitative Protein Expression and Modification in Mammals) will be applied to create a brain atlas of proteomic changes that occur in nicotine-dependent rodents. Tobacco usage remains one the most prevalent forms of substance abuse and exerts a tremendous cost on world health and economy. While it is known that nicotine is the major component in tobacco smoke responsible for addiction, we currently have only a limited understanding of the biochemical alterations caused by nicotine in the nervous system. This project will yield the first large-scale anatomical inventory of nicotine- induced changes in protein expression and post-translational modification in the mammalian brain. These changes will identify key protein pathways that are dysregulated by nicotine, which should in turn serve as fertile ground for future studies aimed at deciphering the neurochemical basis for nicotine dependence. In this way, these proteomic studies will serve as a powerful hypothesis-generating engine that can be leveraged many times over to spawn new basic and translational research programs aimed at understanding and eventually treating nicotine addiction. More generally, this project will deliver a fully integrated and portable quantitative proteomic platform that can be adopted by biological researchers around the world to accelerate the growth and pace of scientific discovery. PUBLIC HEALTH RELEVANCE: Tobacco usage remains one of the most prevalent forms of substance abuse and exerts a tremendous cost on world health and economy. Here, we propose to complete the first large-scale anatomical inventory of nicotine-induced changes in protein expression and post-translational modification in the mammalian brain. These studies should yield new hypotheses to explain the mechanistic basis for nicotine addiction and, through doing so, reveal novel diagnostic and therapeutic strategies to treat this debilitating disease.